Project Summary/Abstract Resistance to the androgen receptor (AR) antagonist enzalutamide remains a major cause of mortality among prostate cancer (PCa) patients. Thus, it is crucial that we identify which patients will become resistant and define the mechanism by which resistance develops. Because metabolic alterations are critical for determining whether cancer cells will resist stress and proliferate, this proposal aims to define the metabolic program that drives enzalutamide resistance. Comparative metabolic studies in six paired enzalutamide-sensitive (EnzS) and enzalutamide-resistant (EnzR) PCa cell lines indicate that EnzR cells have enhanced glutamine metabolism and are more glutamine dependent. This is supported by previous data showing that AR promotes glutamine uptake and glutaminolysis. Further characterization indicates that resistant cells have increased oxidative stress, and the growth of EnzR cells is promoted by antioxidants, such as glutathione (GSH). The role of oxidative stress is corroborated by data suggesting that enzalutamide inhibits antioxidants and induces reactive oxygen species (ROS). Given that an important role of glutamine is to generate antioxidants, we propose that enhanced glutamine metabolism drives enzalutamide resistance in prostate cancer by upregulating antioxidant programs to tolerate increased oxidative stress, and this resistance mechanism is targetable. This proposal will test this hypothesis through the use of unbiased steady-state and metabolic flux techniques in EnzS and EnzR cells in vitro, mouse xenografts in vivo, and patient tumors. The link between metabolic dependencies and ROS will be further characterized by fluorescence microscopy and enzymatic activity approaches. RNAi, CRISPR-Cas9, and lentiviral-mediated overexpression of glutaminase (GLS), a critical enzyme in glutaminolysis, and glutamate-cysteine ligase, a rate-limiting enzyme for GSH synthesis, will be used to establish the necessity and sufficiency of these alterations for driving resistance. Known inhibitors of these enzymes will be used in vitro, in vivo, and in patient-derived tumors cultured ex vivo to determine whether these therapeutic strategies will be potentially efficacious in the treatment of EnzR PCa. This proposed research will provide the understanding of mechanisms that enable the evolution of enzalutamide resistance and allow for the development of therapies leading to more durable responses to enzalutamide in the clinic.